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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure Lindsay A. Starkey a, Anne W. Barrett a, Ramaswamy Chandrashekar b, Brett A. Stillman b, Phyllis Tyrrell b, Brendon Thatcher b, Melissa J. Beall b, Jeff M. Gruntmeir a, James H. Meinkoth a, Susan E. Little a,* a Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078, USA b IDEXX Laboratories, Inc., Westbrook, ME, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2014 Received in revised form 30 July 2014
Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP1 4Dx1 and SNAP1 4Dx1 Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer = 128–4096, GMTMAX = 548.7) and E. chaffeensis (10/10, maximum inverse titer = 1024–32,768, GMTMAX = 4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens. ß 2014 Elsevier B.V. All rights reserved.
Accepted 1 August 2014 Keywords: Dog Ehrlichia chaffeensis Ehrlichia ewingii Ehrlichiosis Ticks
1. Introduction Dogs are known to be susceptible to infection with several different Ehrlichia spp. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, is considered the most pathogenic; in some cases, fatalities result. Ehrlichia ewingii has the capacity to set up long-term infections in
* Corresponding author. Tel.: +1 405 744 8523; fax: +1 405 744 5275. E-mail address: [email protected] (S.E. Little).
dogs and may induce polyarthritis (Little, 2010). Other Ehrlichia spp., including E. chaffeensis, E. muris, and Panola Mountain Ehrlichia (PME), have also been reported from dogs (Little et al., 2010; Hegarty et al., 2012; Qurollo et al., 2013). A number of Ehrlichia spp. also have been reported to cause disease in humans, although E. chaffeensis is considered to be the most common and clinically severe (Nicholson et al., 2010). Amblyomma americanum is responsible for transmission of E. chaffeensis, E. ewingii, and PME (Anziani et al., 1990; Ewing et al., 1995; Yabsley et al., 2008) in the
http://dx.doi.org/10.1016/j.vetmic.2014.08.006 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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southeastern US while Ixodes scapularis is the proposed vector for E. muris-like agents (Pritt et al., 2011; Hegarty et al., 2012). Infection with E. canis is most often seen in dogs because the primary vector, Rhipicephalus sanguineus, prefers to feed on canine hosts (Dantas-Torres, 2010). However, Dermacentor variabilis has also been shown to be capable of transmitting E. canis (Johnson et al., 1998). Dogs in the southeastern United States have the highest seroprevalence for Ehrlichia spp. (Bowman et al., 2009; Beall et al., 2012). Infections are particularly common in areas where A. americanum populations are intense. All of the tick vectors mentioned are present in the southeastern US and may infest dogs in this region (Centers for Disease Control and Prevention, 2014). To determine the risk of ehrlichial infection to dogs, we exposed dogs to tick habitat via weekly walks and evaluated them using clinical, serological, and molecular diagnostic techniques. 2. Materials and methods 2.1. Study participants and pre-screening Oklahoma State University’s Institutional Animal Care and Use Committee reviewed and approved all animal protocols prior to the initiation of this study. Class A Beagle dogs (n = 10), five months of age, were tested for evidence of current or previous infection with E. canis, E. chaffeensis, and E. ewingii by patient-side ELISA, indirect fluorescence antibody (IFA), species-specific peptide analysis, and nested and real-time polymerase chain reaction as described below prior to inclusion in the study. In addition, dogs were screened for infection with Anaplasma spp. and Borrelia burgdorferi (SNAP1 4Dx1), as well as Rickettsia spp. (IFA) as previously described (Barrett et al., 2014). 2.2. Tick exposure and clinical monitoring for disease To ensure exposure to ticks, dogs were walked once per week for seven weeks in May and June, 2011, at a field station in Payne County, Oklahoma, as previously described (Barrett et al., 2014). Ticks acquired were allowed to feed to repletion. After the last walk, ticks were allowed to feed for one additional week and then all of the ticks were removed from each dog. Dogs were monitored daily over the entire exposure period, and for two months following final tick exposure, for clinical signs of infection, including rectal temperature, activity level, myalgia, and ocular discharge. 2.3. Sample collection Prior to tick exposure and twice weekly throughout the 121 day study, whole blood and serum were collected via jugular venipuncture as previously described (Barrett et al., 2014). Blood smears were made weekly. Serum and whole blood were stored at 20 8C until serology or PCR were performed. 2.4. Serology Antibodies to Ehrlichia spp. were detected using IFA tests as previously described (Ristic et al., 1972). Commercially
available slides were used to test sera for antibodies reactive to E. chaffeensis and E. canis (Fuller Laboratories, Fullerton, California) with FITC-labeled goat-anti-dog IgG (KPL, Gaithersburg, Maryland) used to detect bound antibody. Every sample was screened at a 1/128 dilution; serial, twofold dilutions of positive samples were evaluated until fluorescence was no longer observed, and the highest dilution at which specific fluorescence was observed reported as the maximum titer. Species-specific peptide analysis was used to detect antibodies specific for Anaplasma spp. (eenz1), A. phagocytophilum (p44 aph), A. platys (p44 apl), Borrelia burgdorferi (C6), Ehrlichia spp. (p30/p30-1), E. canis (p16), E. chaffeensis (VLPT), and E. ewingii (p28) using a research SNAP prototype (Qurollo et al., 2014). Positive peptide values were quantified using a densigraph to determine the SNAP spot intensity (Chandrashekar et al., 2010). Commercially available patient-side enzyme linked immunosorbent assays (SNAP1 4Dx1 and SNAP1 4Dx Plus1, IDEXX1 Laboratories, Westbrook, Maine) were also employed for detection of antibodies to Ehrlichia spp., Anaplasma spp., and B. burgdorferi. Serum was tested according to manufacturer’s instructions. 2.5. Nested PCR and sequencing DNA was extracted from 200 mL of anticoagulated whole blood with the IllustraTM blood genomic Prep Mini Spin Kit (GE Healthcare UK Limited, Buckinghamshire, UK) according to manufacturer’s instructions from each dog on each collection date, and nucleic acid eluted into a final volume of 200 mL. Separate, dedicated laboratory areas were used for DNA extraction, primary amplification, secondary amplification, and product analyses, and negative (water) controls were included in each extraction and amplification. Species-specific 16S rDNA fragments were amplified by nested PCR using primers ECC/ECB followed by ECA/HE3 (E. canis), HE1/HE3 (E. chaffeensis), and EE72/ HE3 (E. ewingii) as previously described (Little et al., 2010). Starting at study day 51 (d51) and working both toward d0 and d121, consecutive samples were tested until all 10 dogs were negative on two consecutive sample days (1 week). Standard agarose gel electrophoresis was used to confirm presence of amplicons, and representative amplicons purified and concentrated according to manufacturer’s instructions using a commercially available kit (Wizard PCR Preps, Promega Corporation, Madison, WI) were then submitted for sequencing at the Molecular Core Facility at Oklahoma State University (Stillwater, Oklahoma). Resultant sequences were compared to those available in the National Center for Biotechnology Information database, including E. chaffeensis (NR_074500) and E. ewingii (NR_044747). 2.6. Real-time PCR To externally validate the nested PCR results, real-time Ehrlichia spp. PCR was performed at a separate facility using a different approach. Template DNA was extracted from 200 mL canine whole blood using the High Pure PCR Template Preparation Kit (Roche Applied Science, Indianapolis, Indiana)
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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according the manufacturer’s instructions. A final volume of 200 mL of eluted DNA was obtained for each sample. All samples DNA were stored at 20 8C until testing. Real-time PCR hybridization probe assays detecting the disulfide oxidoreductase gene of E. ewingii (DQ902688) and E. chaffeensis (AF403711) were used for the testing of the sample DNA. The real-time PCR assays were performed with the LightCycler 480 instrument (Roche Applied Science, Indianapolis, Indiana). PCR was carried out in a total reaction volume of 20 ml containing LightCycler 480 Genotyping Master mix (Roche Applied Science), species specific primers and probes and 5 ml of template DNA (Ndip et al., 2007). Cycling parameters for the real-time PCR consisted of a denaturation cycle of 95 8C for 10 min, followed by a 55 cycle amplification profile (95 8C for 20 s, 60 8C for 30 s with a single data acquisition, 72 8C for 20 s), a melting curve profile (95 8C for 1 min, 45 8C for 1 min and 80 8C continuous with a ramp rate of 0.14 8C per second and 4 data acquisitions per 8C) and a cool cycle of 40 8C for 30 s. In each run, 105 and 102 copies of recombinant plasmids containing an insert of the species specific target were tested as positive controls. PCR grade water (Roche Applied Science, Indianapolis, Indiana) was tested as the negative control. Analytical sensitivity was determined to be 10 gene copies using the assay specific plasmids. 2.7. Complete blood counts and blood smears Whole blood was submitted for complete blood count to the clinical pathology laboratory service, Oklahoma State University, from each dog on days 16, 44, 58, 72, and 86. Thin blood smears were air dried, fixed in methanol, stained using Wright’s–Giemsa, and then examined microscopically for morulae within leukocytes by a boarded clinical pathologist (JHM). 3. Results All dogs used in this study were seronegative for antibodies reactive to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. on all methods used, as well as PCR negative for Ehrlichia spp., prior to natural tick exposure. As previously reported, dogs were infested with ticks on every exposure date for a total infestation of 57–108 ticks per dog (Barrett et al., 2014). The majority of ticks present were
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A. americanum, however low numbers of D. variabilis and A. maculatum were also seen. Clinical signs of illness were never observed in any dog (Barrett et al., 2014). All 10 dogs developed antibody titers on IFA to both E. canis and E. chaffeensis (Table 1). Maximum inverse titers ranged from 128 to 4096 for E. canis and 1024 to 32,768 for E. chaffeensis. The geometric mean of the maximum inverse titers (GMTMAX) for E. canis and E. chaffeensis were 548.7 and 4096, respectively (Fig. 1). Species-specific peptide analysis using the microtiter well based assays revealed antibodies to E. chaffeensis (VLPT) in 7/10 dogs, and E. ewingii (p28) in 9/10 dogs. Positive peptide values ranged from 0.03 to 0.45 for E. chaffeensis and 0.04 to 0.85 for E. ewingii (Fig. 1). Antibodies to E. canis (p16), B. burgdorferi (C6), or Anaplasma spp. (eenz1, p44 aph, p44 apl) were not detected (Table 1). On d121, 4/10 dogs had antibodies reactive to Ehrlichia spp. (p30/30-1) using the SNAP1 4Dx1 patient-side ELISA, and 7/10 had antibodies reactive to Ehrlichia spp. (p30/30-1 and p28) using the SNAP1 4Dx1 Plus (IDEXX Laboratories Inc., Westbrook, Maine). The three dogs which were negative on d121 on SNAP1 4Dx1 Plus were positive on d61 or d93; in total, 10/10 dogs developed antibodies reactive to Ehrlichia spp. on SNAP1 4Dx1 Plus during the study. On nested PCR, 7/10 dogs tested positive on at least one study date for the presence of E. chaffeensis (Table 2a), 10/ 10 for E. ewingii (Table 2b), and 0/10 for E. canis (data not shown). Sequences from representative amplicons aligned with 100% identity to those for E. chaffeensis (NR_074500) or E. ewingii (NR_044747). Rickettsemia with E. chaffeensis and E. ewingii was detected intermittently in blood via nested PCR for an average of 3.2 and 30.5 total days, respectively. Real-time PCR detected E. chaffeensis DNA in 0/10 and E. ewingii DNA in 9/10 dogs throughout the study (Tables 2a and 2b). As previously reported (Barrett et al., 2014), no significant changes were observed on any CBC. A single morula was found in a neutrophil from one dog on d61 and in two neutrophils from a second dog on d72. 4. Discussion Diagnosis of tick-borne infections is increasingly common in the United States, both in animals and people (Nicholson et al., 2010). Our data reveal that the risk of
Table 1 Antibodies detected to Ehrlichia canis, E. chaffeensis, E. ewingii, Ehrlichia spp., Anaplasma platys, A. phagocytophilum, and Borrelia burgdorferi in 10 dogs naturally infested with ticks. Organism
18 Tick vector
Analyte
E. canis E. chaffeensis Ehrlichia spp. E. canis E. chaffeensis E. ewingii B. burgdorferi Anaplasma spp. A. phagocytophilum A. platys
R. sanguineus A. americanum Various R. sanguineus A. americanum A. americanum I. scapularis Various I. scapularis R. sanguineus
IFA IFA p30/p30-1 p16 VLPT p28 C6 eenz1 p44 aph p44 apl
a
Months after initial tick infestationa 0
1
2
3
4
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
1/10 5/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10 0/10
8/10 9/10 6/10 0/10 5/10 6/10 0/10 0/10 0/10 0/10
8/10 9/10 6/10 0/10 6/10 8/10 0/10 0/10 0/10 0/10
8/10 8/10 7/10 0/10 2/10 8/10 0/10 0/10 0/10 0/10
Serum was tested by IFA on study days 30, 61, 93, and 121 and by ELISA using specific peptides on study days 33, 65, 89, and 117.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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Fig. 1. Geometric mean titers to Ehrlichia chaffeensis and Ehrlichia canis on IFA (lines with standard errors) and arithmetic mean peptide values to Ehrlichia ewingii/p28 and E. chaffeensis/VLPT (bars with standard errors) in dogs naturally infested with ticks. Antibodies to E. canis/p16 were not detected in any dog.
following natural tick exposure in the present study supports dogs as the proposed reservoir host for E. ewingii (Yabsley et al., 2011), although further study is needed to confirm that interpretation. Results of the various diagnostic assays used in these dogs largely agreed with one another, although nonspecific antibodies were detected on IFA before the specific peptide-based assays (Fig. 1), and peptide-based assays may detect antibodies against Ehrlichia spp. that have not yet been described (Little, 2010). In addition, occasional discordant PCR results were observed which could be due to the different targets used, the relative sensitivity of the
infection is high even with a relatively limited time period of tick exposure. As evidenced by PCR and serologic data, all dogs in this study became infected with Ehrlichia sp(p)., although infection with E. ewingii was more common than infection with E. chaffeensis. Some dogs remained PCR positive intermittently throughout the study, with 4/10 dogs still PCR positive for E. ewingii on d121, over two months after the final exposure to ticks, indicating persistent infection had been established. Previous work has shown that dogs can become persistently infected with E. ewingii following intravenous inoculation (Yabsley et al., 2011). The finding of persistent E. ewingii rickettsemia
Table 2a Detection by nested (and real-time) PCR of Ehrlichia chaffeensis in 10 dogs naturally infested with ticks. Study day
Dog number 2
1 13 16 19 23 26 30 33 37 40 44 47 51 55 59 61 65 67 72 75 Totala a
( )
3
4
5
6
7
8
9
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
+ +( )
( )
10 ( )
( )
( )
+( )
+
( )
+
( )
+ + + +( ) +
+
( )
( )
( )
( )
+
( )
( )
+ +( )
( )
( )
( )
+ ( )
( )
( )
+( ) +
( )
( )
+ ( )
( )
+
0
16
0
1
1
0
8
3
1
2
Total number of consecutive days, inclusive, for which E. chaffeensis was detected by PCR.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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Table 2b Detection by nested (and real-time) PCR of Ehrlichia ewingii in 10 dogs naturally infested with ticks. Study day
Dog number 2
1 30 33 37 40 44 47 51 55 59 61 65 67 72 75 79 82 86 89 93 96 100 103 107 110 114 117 121 Totala a
( )
( ) + + + + (+) + + + (+)
( )
3 ( )
+ (+) + + + +( ) +
4 ( )
+ + (+) + + + ( )
( )
( )
( )
( )
5 ( )
6 ( )
( )
( )
+ ( )
( )
+ + + (+) + + + (+)
( )
( )
+ + + (+)
( ) 23
+(+) + + + + (+) + 34
( )
( ) 14
( )
( )
( ) 17
( ) 1
7 ( )
( ) + + + (+) + + + + (+) + + + (+) + + + +( ) + + + +( ) + 62
8 ( )
+ +( ) + + + (+)
( )
( )
+ (+) + + + + (+) + 31
9 ( )
+ + + + + + + + + + + + + + + +
(+)
10 ( )
( )
(+)
+( ) + + + + (+) + + + ( )
(+)
( )
(+)
(+)
+ (+) + + + + (+) + +
( )
(+)
( )
(+)
( )
+
53
21
+ + + (+) + + + + (+) + 49
Total number of consecutive days, inclusive, for which E. ewingii was detected by nested PCR.
different assays, lower amounts of target present, or loss of detectable nucleic acid during storage of samples (Allison and Little, 2013). When considered together, the data from the present study support using multiple diagnostic modalities to identify infection, particularly early in infection when disease is most likely to develop (Little, 2010). However, identifying the most reliable testing modality for detecting early infection requires further exploration. Even though all dogs were shown to be infected with one or more Ehrlichia spp., none of the dogs exhibited clinical signs of illness, nor were there any blood work abnormalities indicative of infection with an Ehrlichia spp. This observation mirrors what is commonly seen by practicing veterinarians: antibodies to one or more Ehrlichia spp. are commonly detected in dogs in which clinical disease is absent or inapparent (Little et al., 2010). Compared to E. canis, infection with E. chaffeensis is thought to be less pathogenic in dogs (Little, 2010), and disease from E. ewingii varies, with only a portion of infected dogs developing clinical illness (Anziani et al., 1990). Interestingly, the dogs in the present study were also infected with Rickettsia spp., albeit of unknown pathogenicity (Barrett et al., 2014). Despite this coinfection, disease was not evident. Previous work has shown co-infection with E. canis and A. platys does result in more severe clinical disease in dogs than either agent alone (Gaunt et al., 2010). Co-infection with multiple tick-borne disease agents has been reported in naturally infected dogs, although the influence of co-infection on disease
severity is not always clear (Kordick et al., 1999; Little et al., 2010). Results from the present study show that dogs are a sensitive indicator for the presence of Ehrlichia infections in ticks; all ten dogs naturally exposed to ticks became infected with at least one Ehrlichia species in a single transmission season (Table 1). Canine seroprevalence studies have shown that antibodies to Ehrlichia spp. are most common in dogs from southeastern United States, with some areas identified where more than 10% of dogs test positive (Murphy et al., 1998; Bowman et al., 2009). In hyperendemic areas of Arkansas and Missouri, prevalence may be even higher; as many as 36.9% of dogs had antibodies to E. chaffeensis and 21.4% to E. ewingii (Beall et al., 2012). Our data suggest that wide scale studies using canine serology may provide valuable insights into risk for human ehrlichiosis, similar to the success seen with this approach in understanding the risk of other tick-borne disease agents, such as Borrelia burgdorferi (Duncan et al., 2005).
Conflict of interest In the past five years, SL and JM have received honoraria and/or research support from IDEXX Laboratories, Inc., a company that manufactures some of the assays used in this research. Salary support for LS is provided through funding from Bayer Animal Health, a manufacturer of tick control products for pets. In addition, RC, BS, PT, BT, and MB are
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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employees of IDEXX Laboratories, Inc. The other authors have no conflicts of interest to report. Acknowledgements Funding to support collection of the samples used in this research was provided by Bayer Animal Health. Dr. Starkey is the Bayer Resident in Veterinary Parasitology at the National Center for Veterinary Parasitology, Oklahoma State University, which provides her salary. Many thanks to technical staff at IDEXX Laboratories, Inc. for assistance with serologic and molecular testing, and laboratory animal resources at Oklahoma State University for support and care of the animals involved in this study. References Allison, R.W., Little, S.E., 2013. Diagnosis of rickettsial diseases in dogs and cats. Vet. Clin. Pathol. 42, 127–144. Anziani, D.S., Ewing, S.A., Barker, R.W., 1990. Experimental transmission of a granulocytic form of the tribe Ehrlichieae by Dermacentor variabilis and Amblyomma americanum to dogs. Am. J. Vet. Res. 51, 929–931. Barrett, A., Little, S.E., Shaw, E., 2014. Rickettsia amblyommii and R. montanensis infection in dogs following natural exposure to ticks. Vector Borne Zoonotic Dis. 14, 20–25. Beall, M.J., Alleman, A.R., Breitschwerdt, E.B., Cohn, L.A., Couto, C.G., Dryden, M.W., Guptill, L.C., Iazbik, C., Kania, S.A., Lathan, P., Little, S.E., Roy, A., Sayler, K.A., Stillman, B.A., Welles, E.G., Wolfson, W., Yabsley, M.J., 2012. Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii in dogs in North America. Parasit. Vectors 5, 29. Bowman, D., Little, S.E., Lorentzen, L., Shields, J., Sullivan, M.P., Carlin, E.P., 2009. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet. Parasitol. 160, 138–148. Centers for Disease Control and Prevention, 2014. http://www.cdc.gov/ ticks/geographic_distribution.html (accessed 06.24.14). Chandrashekar, R., Mainville, C.A., Beall, M.J., O’Connor, T., Eberts, M.D., Alleman, A.R., Gaunt, S.D., Breitschwerdt, E.B., 2010. Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs. Am. J. Vet. Res. 71, 1443–1450. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 3, 26. Duncan, A.W., Correa, M.T., Levine, J.F., Breitschwerdt, E.B., 2005. The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic states. Vector Borne Zoonotic Dis. 5, 101–109. Ewing, S.A., Dawson, J.E., Kocan, A.A., Barker, R.W., Warner, C.K., Panciera, R.J., Fox, J.C., Kocan, K.M., Blouin, E.F., 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-
tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32, 368–374. Gaunt, S., Beall, M., Stillman, B., Lorentzen, L., Diniz, P., Chandrashekar, R., Breitschwerdt, E., 2010. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic, and molecular findings. Parasit. Vectors 3, 33. Hegarty, B.C., Maggi, R.G., Koskinen, P., Beall, M.J., Eberts, M., Chandrashekar, R., Breitschwerdt, E.B., 2012. Ehrlichia muris infection in a dog from Minnesota. J. Vet. Intern. Med. 26, 1217–1220. Johnson, E.M., Ewing, S.A., Barker, R.W., Fox, J.C., Crow, D.W., Kocan, K.M., 1998. Experimental transmission of Ehrlichia canis (Rickettsiales: Ehrllichieae) by Dermacentor variabilis (Acari: Ixodidae). Vet. Parasitol. 74, 277–288. Kordick, S.K., Breitschwerdt, E.B., Hegarty, B.C., Southwick, K.L., Colitz, C.M., Hancock, S.I., Bradley, J.M., Rumbough, R., McPherson, J.T., MacCormack, J.N., 1999. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J. Clin. Microbiol. 37, 2631–2638. Little, S.E., 2010. Ehrlichiosis and anaplasmosis in dogs and cats. Vet. Clin. North Am. Small Anim. Pract. 40, 1121–1140. Little, S.E., O’Connor, T.P., Hempstead, J., Saucier, J., Reichard, M.V., Meinkoth, K., Meinkoth, J.H., Andrews, B., Ullom, S., Ewing, S.A., Chandrashekar, R., 2010. Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States. Vet. Parasitol. 172, 355–360. Murphy, G.L., Ewing, S.A., Whitworth, L.C., Fox, J.C., Kocan, A.A., 1998. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol. 79, 325–339. Ndip, L.M., Ndip, R.N., Ndive, V.E., Awuh, J.A., Walker, D.H., McBride, J.W., 2007. Ehrlichia species in Rhipicephalus sanguineus ticks in Cameroon. Vector Borne Zoonotic Dis. 7, 221–227. Nicholson, W.L., Allen, K.E., McQuiston, J.H., Breitschwerdt, E.B., Little, S.E., 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol. 26, 205–212. Pritt, B.S., Sloan, L.M., Johnson, D.K., Munderloh, U.G., Paskewitz, S.M., McElroy, K.M., McFadden, J.D., Binnicker, M.J., Neitzel, D.F., Liu, G., Nicholson, W.L., Nelson, C.M., Franson, J.J., Martin, S.A., Cunningham, S.A., Steward, C.R., Bogumill, K., Bjorgaard, M.E., Davis, J.P., McQuiston, J.H., Warshauer, D.M., Wilhelm, M.P., Patel, R., Trivedi, V.A., Eremeeva, M.E., 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365, 422–429. Qurollo, B.A., Davenport, A.C., Sherbert, B.M., Grindem, C.B., Birkenheuer, A.J., Breitschwerdt, E.B., 2013. Infection with Panola Mountain Ehrlichia sp. in a dog with atypical lymphocytes and clonal T-cell expansion. J. Vet. Intern. Med. 27, 1251–1255. Qurollo, B.A., Chandrashekar, R., Hegarty, B.C., Beall, M.J., Stillman, B., Liu, J., Thatcher, B., Pultorak, B., Comyn, A., Bretischwerdt, E.B., 2014. A descriptive analysis of clinical manifestations in dogs with tick-borne pathogen co-infections and co-exposures. J. Vet. Intern. Med. 28, 976–1134. Ristic, M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K., Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun. 6, 226–231. Yabsley, M.J., Adams, D.S., O’Connor, T.P., Chandrashekar, R., Little, S.E., 2011. Experimental primary and secondary infections of domestic dogs with Ehrlichia ewingii. Vet. Microbiol. 150, 315–321. Yabsley, M.J., Loftis, A.D., Little, S.E., 2008. Natural and experimental infection of white-tailed deer (Odocoileus virginianus) from the United States with an Ehrlichia sp. closely related to Ehrlichia ruminantium. J. Wildl. Dis. 44, 381–387.
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VETMIC-6702; No. of Pages 6 Veterinary Microbiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short Communication
Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure Lindsay A. Starkey a, Anne W. Barrett a, Ramaswamy Chandrashekar b, Brett A. Stillman b, Phyllis Tyrrell b, Brendon Thatcher b, Melissa J. Beall b, Jeff M. Gruntmeir a, James H. Meinkoth a, Susan E. Little a,* a Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, 250 McElroy Hall, Stillwater, OK 74078, USA b IDEXX Laboratories, Inc., Westbrook, ME, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 March 2014 Received in revised form 30 July 2014
Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP1 4Dx1 and SNAP1 4Dx1 Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichia canis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer = 128–4096, GMTMAX = 548.7) and E. chaffeensis (10/10, maximum inverse titer = 1024–32,768, GMTMAX = 4096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens. ß 2014 Elsevier B.V. All rights reserved.
Accepted 1 August 2014 Keywords: Dog Ehrlichia chaffeensis Ehrlichia ewingii Ehrlichiosis Ticks
1. Introduction Dogs are known to be susceptible to infection with several different Ehrlichia spp. Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, is considered the most pathogenic; in some cases, fatalities result. Ehrlichia ewingii has the capacity to set up long-term infections in
* Corresponding author. Tel.: +1 405 744 8523; fax: +1 405 744 5275. E-mail address: [email protected] (S.E. Little).
dogs and may induce polyarthritis (Little, 2010). Other Ehrlichia spp., including E. chaffeensis, E. muris, and Panola Mountain Ehrlichia (PME), have also been reported from dogs (Little et al., 2010; Hegarty et al., 2012; Qurollo et al., 2013). A number of Ehrlichia spp. also have been reported to cause disease in humans, although E. chaffeensis is considered to be the most common and clinically severe (Nicholson et al., 2010). Amblyomma americanum is responsible for transmission of E. chaffeensis, E. ewingii, and PME (Anziani et al., 1990; Ewing et al., 1995; Yabsley et al., 2008) in the
http://dx.doi.org/10.1016/j.vetmic.2014.08.006 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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southeastern US while Ixodes scapularis is the proposed vector for E. muris-like agents (Pritt et al., 2011; Hegarty et al., 2012). Infection with E. canis is most often seen in dogs because the primary vector, Rhipicephalus sanguineus, prefers to feed on canine hosts (Dantas-Torres, 2010). However, Dermacentor variabilis has also been shown to be capable of transmitting E. canis (Johnson et al., 1998). Dogs in the southeastern United States have the highest seroprevalence for Ehrlichia spp. (Bowman et al., 2009; Beall et al., 2012). Infections are particularly common in areas where A. americanum populations are intense. All of the tick vectors mentioned are present in the southeastern US and may infest dogs in this region (Centers for Disease Control and Prevention, 2014). To determine the risk of ehrlichial infection to dogs, we exposed dogs to tick habitat via weekly walks and evaluated them using clinical, serological, and molecular diagnostic techniques. 2. Materials and methods 2.1. Study participants and pre-screening Oklahoma State University’s Institutional Animal Care and Use Committee reviewed and approved all animal protocols prior to the initiation of this study. Class A Beagle dogs (n = 10), five months of age, were tested for evidence of current or previous infection with E. canis, E. chaffeensis, and E. ewingii by patient-side ELISA, indirect fluorescence antibody (IFA), species-specific peptide analysis, and nested and real-time polymerase chain reaction as described below prior to inclusion in the study. In addition, dogs were screened for infection with Anaplasma spp. and Borrelia burgdorferi (SNAP1 4Dx1), as well as Rickettsia spp. (IFA) as previously described (Barrett et al., 2014). 2.2. Tick exposure and clinical monitoring for disease To ensure exposure to ticks, dogs were walked once per week for seven weeks in May and June, 2011, at a field station in Payne County, Oklahoma, as previously described (Barrett et al., 2014). Ticks acquired were allowed to feed to repletion. After the last walk, ticks were allowed to feed for one additional week and then all of the ticks were removed from each dog. Dogs were monitored daily over the entire exposure period, and for two months following final tick exposure, for clinical signs of infection, including rectal temperature, activity level, myalgia, and ocular discharge. 2.3. Sample collection Prior to tick exposure and twice weekly throughout the 121 day study, whole blood and serum were collected via jugular venipuncture as previously described (Barrett et al., 2014). Blood smears were made weekly. Serum and whole blood were stored at 20 8C until serology or PCR were performed. 2.4. Serology Antibodies to Ehrlichia spp. were detected using IFA tests as previously described (Ristic et al., 1972). Commercially
available slides were used to test sera for antibodies reactive to E. chaffeensis and E. canis (Fuller Laboratories, Fullerton, California) with FITC-labeled goat-anti-dog IgG (KPL, Gaithersburg, Maryland) used to detect bound antibody. Every sample was screened at a 1/128 dilution; serial, twofold dilutions of positive samples were evaluated until fluorescence was no longer observed, and the highest dilution at which specific fluorescence was observed reported as the maximum titer. Species-specific peptide analysis was used to detect antibodies specific for Anaplasma spp. (eenz1), A. phagocytophilum (p44 aph), A. platys (p44 apl), Borrelia burgdorferi (C6), Ehrlichia spp. (p30/p30-1), E. canis (p16), E. chaffeensis (VLPT), and E. ewingii (p28) using a research SNAP prototype (Qurollo et al., 2014). Positive peptide values were quantified using a densigraph to determine the SNAP spot intensity (Chandrashekar et al., 2010). Commercially available patient-side enzyme linked immunosorbent assays (SNAP1 4Dx1 and SNAP1 4Dx Plus1, IDEXX1 Laboratories, Westbrook, Maine) were also employed for detection of antibodies to Ehrlichia spp., Anaplasma spp., and B. burgdorferi. Serum was tested according to manufacturer’s instructions. 2.5. Nested PCR and sequencing DNA was extracted from 200 mL of anticoagulated whole blood with the IllustraTM blood genomic Prep Mini Spin Kit (GE Healthcare UK Limited, Buckinghamshire, UK) according to manufacturer’s instructions from each dog on each collection date, and nucleic acid eluted into a final volume of 200 mL. Separate, dedicated laboratory areas were used for DNA extraction, primary amplification, secondary amplification, and product analyses, and negative (water) controls were included in each extraction and amplification. Species-specific 16S rDNA fragments were amplified by nested PCR using primers ECC/ECB followed by ECA/HE3 (E. canis), HE1/HE3 (E. chaffeensis), and EE72/ HE3 (E. ewingii) as previously described (Little et al., 2010). Starting at study day 51 (d51) and working both toward d0 and d121, consecutive samples were tested until all 10 dogs were negative on two consecutive sample days (1 week). Standard agarose gel electrophoresis was used to confirm presence of amplicons, and representative amplicons purified and concentrated according to manufacturer’s instructions using a commercially available kit (Wizard PCR Preps, Promega Corporation, Madison, WI) were then submitted for sequencing at the Molecular Core Facility at Oklahoma State University (Stillwater, Oklahoma). Resultant sequences were compared to those available in the National Center for Biotechnology Information database, including E. chaffeensis (NR_074500) and E. ewingii (NR_044747). 2.6. Real-time PCR To externally validate the nested PCR results, real-time Ehrlichia spp. PCR was performed at a separate facility using a different approach. Template DNA was extracted from 200 mL canine whole blood using the High Pure PCR Template Preparation Kit (Roche Applied Science, Indianapolis, Indiana)
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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according the manufacturer’s instructions. A final volume of 200 mL of eluted DNA was obtained for each sample. All samples DNA were stored at 20 8C until testing. Real-time PCR hybridization probe assays detecting the disulfide oxidoreductase gene of E. ewingii (DQ902688) and E. chaffeensis (AF403711) were used for the testing of the sample DNA. The real-time PCR assays were performed with the LightCycler 480 instrument (Roche Applied Science, Indianapolis, Indiana). PCR was carried out in a total reaction volume of 20 ml containing LightCycler 480 Genotyping Master mix (Roche Applied Science), species specific primers and probes and 5 ml of template DNA (Ndip et al., 2007). Cycling parameters for the real-time PCR consisted of a denaturation cycle of 95 8C for 10 min, followed by a 55 cycle amplification profile (95 8C for 20 s, 60 8C for 30 s with a single data acquisition, 72 8C for 20 s), a melting curve profile (95 8C for 1 min, 45 8C for 1 min and 80 8C continuous with a ramp rate of 0.14 8C per second and 4 data acquisitions per 8C) and a cool cycle of 40 8C for 30 s. In each run, 105 and 102 copies of recombinant plasmids containing an insert of the species specific target were tested as positive controls. PCR grade water (Roche Applied Science, Indianapolis, Indiana) was tested as the negative control. Analytical sensitivity was determined to be 10 gene copies using the assay specific plasmids. 2.7. Complete blood counts and blood smears Whole blood was submitted for complete blood count to the clinical pathology laboratory service, Oklahoma State University, from each dog on days 16, 44, 58, 72, and 86. Thin blood smears were air dried, fixed in methanol, stained using Wright’s–Giemsa, and then examined microscopically for morulae within leukocytes by a boarded clinical pathologist (JHM). 3. Results All dogs used in this study were seronegative for antibodies reactive to Anaplasma spp., Borrelia burgdorferi, and Ehrlichia spp. on all methods used, as well as PCR negative for Ehrlichia spp., prior to natural tick exposure. As previously reported, dogs were infested with ticks on every exposure date for a total infestation of 57–108 ticks per dog (Barrett et al., 2014). The majority of ticks present were
3
A. americanum, however low numbers of D. variabilis and A. maculatum were also seen. Clinical signs of illness were never observed in any dog (Barrett et al., 2014). All 10 dogs developed antibody titers on IFA to both E. canis and E. chaffeensis (Table 1). Maximum inverse titers ranged from 128 to 4096 for E. canis and 1024 to 32,768 for E. chaffeensis. The geometric mean of the maximum inverse titers (GMTMAX) for E. canis and E. chaffeensis were 548.7 and 4096, respectively (Fig. 1). Species-specific peptide analysis using the microtiter well based assays revealed antibodies to E. chaffeensis (VLPT) in 7/10 dogs, and E. ewingii (p28) in 9/10 dogs. Positive peptide values ranged from 0.03 to 0.45 for E. chaffeensis and 0.04 to 0.85 for E. ewingii (Fig. 1). Antibodies to E. canis (p16), B. burgdorferi (C6), or Anaplasma spp. (eenz1, p44 aph, p44 apl) were not detected (Table 1). On d121, 4/10 dogs had antibodies reactive to Ehrlichia spp. (p30/30-1) using the SNAP1 4Dx1 patient-side ELISA, and 7/10 had antibodies reactive to Ehrlichia spp. (p30/30-1 and p28) using the SNAP1 4Dx1 Plus (IDEXX Laboratories Inc., Westbrook, Maine). The three dogs which were negative on d121 on SNAP1 4Dx1 Plus were positive on d61 or d93; in total, 10/10 dogs developed antibodies reactive to Ehrlichia spp. on SNAP1 4Dx1 Plus during the study. On nested PCR, 7/10 dogs tested positive on at least one study date for the presence of E. chaffeensis (Table 2a), 10/ 10 for E. ewingii (Table 2b), and 0/10 for E. canis (data not shown). Sequences from representative amplicons aligned with 100% identity to those for E. chaffeensis (NR_074500) or E. ewingii (NR_044747). Rickettsemia with E. chaffeensis and E. ewingii was detected intermittently in blood via nested PCR for an average of 3.2 and 30.5 total days, respectively. Real-time PCR detected E. chaffeensis DNA in 0/10 and E. ewingii DNA in 9/10 dogs throughout the study (Tables 2a and 2b). As previously reported (Barrett et al., 2014), no significant changes were observed on any CBC. A single morula was found in a neutrophil from one dog on d61 and in two neutrophils from a second dog on d72. 4. Discussion Diagnosis of tick-borne infections is increasingly common in the United States, both in animals and people (Nicholson et al., 2010). Our data reveal that the risk of
Table 1 Antibodies detected to Ehrlichia canis, E. chaffeensis, E. ewingii, Ehrlichia spp., Anaplasma platys, A. phagocytophilum, and Borrelia burgdorferi in 10 dogs naturally infested with ticks. Organism
18 Tick vector
Analyte
E. canis E. chaffeensis Ehrlichia spp. E. canis E. chaffeensis E. ewingii B. burgdorferi Anaplasma spp. A. phagocytophilum A. platys
R. sanguineus A. americanum Various R. sanguineus A. americanum A. americanum I. scapularis Various I. scapularis R. sanguineus
IFA IFA p30/p30-1 p16 VLPT p28 C6 eenz1 p44 aph p44 apl
a
Months after initial tick infestationa 0
1
2
3
4
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10
1/10 5/10 0/10 0/10 1/10 0/10 0/10 0/10 0/10 0/10
8/10 9/10 6/10 0/10 5/10 6/10 0/10 0/10 0/10 0/10
8/10 9/10 6/10 0/10 6/10 8/10 0/10 0/10 0/10 0/10
8/10 8/10 7/10 0/10 2/10 8/10 0/10 0/10 0/10 0/10
Serum was tested by IFA on study days 30, 61, 93, and 121 and by ELISA using specific peptides on study days 33, 65, 89, and 117.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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4
Fig. 1. Geometric mean titers to Ehrlichia chaffeensis and Ehrlichia canis on IFA (lines with standard errors) and arithmetic mean peptide values to Ehrlichia ewingii/p28 and E. chaffeensis/VLPT (bars with standard errors) in dogs naturally infested with ticks. Antibodies to E. canis/p16 were not detected in any dog.
following natural tick exposure in the present study supports dogs as the proposed reservoir host for E. ewingii (Yabsley et al., 2011), although further study is needed to confirm that interpretation. Results of the various diagnostic assays used in these dogs largely agreed with one another, although nonspecific antibodies were detected on IFA before the specific peptide-based assays (Fig. 1), and peptide-based assays may detect antibodies against Ehrlichia spp. that have not yet been described (Little, 2010). In addition, occasional discordant PCR results were observed which could be due to the different targets used, the relative sensitivity of the
infection is high even with a relatively limited time period of tick exposure. As evidenced by PCR and serologic data, all dogs in this study became infected with Ehrlichia sp(p)., although infection with E. ewingii was more common than infection with E. chaffeensis. Some dogs remained PCR positive intermittently throughout the study, with 4/10 dogs still PCR positive for E. ewingii on d121, over two months after the final exposure to ticks, indicating persistent infection had been established. Previous work has shown that dogs can become persistently infected with E. ewingii following intravenous inoculation (Yabsley et al., 2011). The finding of persistent E. ewingii rickettsemia
Table 2a Detection by nested (and real-time) PCR of Ehrlichia chaffeensis in 10 dogs naturally infested with ticks. Study day
Dog number 2
1 13 16 19 23 26 30 33 37 40 44 47 51 55 59 61 65 67 72 75 Totala a
( )
3
4
5
6
7
8
9
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
( )
+ +( )
( )
10 ( )
( )
( )
+( )
+
( )
+
( )
+ + + +( ) +
+
( )
( )
( )
( )
+
( )
( )
+ +( )
( )
( )
( )
+ ( )
( )
( )
+( ) +
( )
( )
+ ( )
( )
+
0
16
0
1
1
0
8
3
1
2
Total number of consecutive days, inclusive, for which E. chaffeensis was detected by PCR.
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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5
Table 2b Detection by nested (and real-time) PCR of Ehrlichia ewingii in 10 dogs naturally infested with ticks. Study day
Dog number 2
1 30 33 37 40 44 47 51 55 59 61 65 67 72 75 79 82 86 89 93 96 100 103 107 110 114 117 121 Totala a
( )
( ) + + + + (+) + + + (+)
( )
3 ( )
+ (+) + + + +( ) +
4 ( )
+ + (+) + + + ( )
( )
( )
( )
( )
5 ( )
6 ( )
( )
( )
+ ( )
( )
+ + + (+) + + + (+)
( )
( )
+ + + (+)
( ) 23
+(+) + + + + (+) + 34
( )
( ) 14
( )
( )
( ) 17
( ) 1
7 ( )
( ) + + + (+) + + + + (+) + + + (+) + + + +( ) + + + +( ) + 62
8 ( )
+ +( ) + + + (+)
( )
( )
+ (+) + + + + (+) + 31
9 ( )
+ + + + + + + + + + + + + + + +
(+)
10 ( )
( )
(+)
+( ) + + + + (+) + + + ( )
(+)
( )
(+)
(+)
+ (+) + + + + (+) + +
( )
(+)
( )
(+)
( )
+
53
21
+ + + (+) + + + + (+) + 49
Total number of consecutive days, inclusive, for which E. ewingii was detected by nested PCR.
different assays, lower amounts of target present, or loss of detectable nucleic acid during storage of samples (Allison and Little, 2013). When considered together, the data from the present study support using multiple diagnostic modalities to identify infection, particularly early in infection when disease is most likely to develop (Little, 2010). However, identifying the most reliable testing modality for detecting early infection requires further exploration. Even though all dogs were shown to be infected with one or more Ehrlichia spp., none of the dogs exhibited clinical signs of illness, nor were there any blood work abnormalities indicative of infection with an Ehrlichia spp. This observation mirrors what is commonly seen by practicing veterinarians: antibodies to one or more Ehrlichia spp. are commonly detected in dogs in which clinical disease is absent or inapparent (Little et al., 2010). Compared to E. canis, infection with E. chaffeensis is thought to be less pathogenic in dogs (Little, 2010), and disease from E. ewingii varies, with only a portion of infected dogs developing clinical illness (Anziani et al., 1990). Interestingly, the dogs in the present study were also infected with Rickettsia spp., albeit of unknown pathogenicity (Barrett et al., 2014). Despite this coinfection, disease was not evident. Previous work has shown co-infection with E. canis and A. platys does result in more severe clinical disease in dogs than either agent alone (Gaunt et al., 2010). Co-infection with multiple tick-borne disease agents has been reported in naturally infected dogs, although the influence of co-infection on disease
severity is not always clear (Kordick et al., 1999; Little et al., 2010). Results from the present study show that dogs are a sensitive indicator for the presence of Ehrlichia infections in ticks; all ten dogs naturally exposed to ticks became infected with at least one Ehrlichia species in a single transmission season (Table 1). Canine seroprevalence studies have shown that antibodies to Ehrlichia spp. are most common in dogs from southeastern United States, with some areas identified where more than 10% of dogs test positive (Murphy et al., 1998; Bowman et al., 2009). In hyperendemic areas of Arkansas and Missouri, prevalence may be even higher; as many as 36.9% of dogs had antibodies to E. chaffeensis and 21.4% to E. ewingii (Beall et al., 2012). Our data suggest that wide scale studies using canine serology may provide valuable insights into risk for human ehrlichiosis, similar to the success seen with this approach in understanding the risk of other tick-borne disease agents, such as Borrelia burgdorferi (Duncan et al., 2005).
Conflict of interest In the past five years, SL and JM have received honoraria and/or research support from IDEXX Laboratories, Inc., a company that manufactures some of the assays used in this research. Salary support for LS is provided through funding from Bayer Animal Health, a manufacturer of tick control products for pets. In addition, RC, BS, PT, BT, and MB are
Please cite this article in press as: Starkey, L.A., et al., Development of antibodies to and PCR detection of Ehrlichia spp. in dogs following natural tick exposure. Vet. Microbiol. (2014), http://dx.doi.org/10.1016/j.vetmic.2014.08.006
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employees of IDEXX Laboratories, Inc. The other authors have no conflicts of interest to report. Acknowledgements Funding to support collection of the samples used in this research was provided by Bayer Animal Health. Dr. Starkey is the Bayer Resident in Veterinary Parasitology at the National Center for Veterinary Parasitology, Oklahoma State University, which provides her salary. Many thanks to technical staff at IDEXX Laboratories, Inc. for assistance with serologic and molecular testing, and laboratory animal resources at Oklahoma State University for support and care of the animals involved in this study. References Allison, R.W., Little, S.E., 2013. Diagnosis of rickettsial diseases in dogs and cats. Vet. Clin. Pathol. 42, 127–144. Anziani, D.S., Ewing, S.A., Barker, R.W., 1990. Experimental transmission of a granulocytic form of the tribe Ehrlichieae by Dermacentor variabilis and Amblyomma americanum to dogs. Am. J. Vet. Res. 51, 929–931. Barrett, A., Little, S.E., Shaw, E., 2014. Rickettsia amblyommii and R. montanensis infection in dogs following natural exposure to ticks. Vector Borne Zoonotic Dis. 14, 20–25. Beall, M.J., Alleman, A.R., Breitschwerdt, E.B., Cohn, L.A., Couto, C.G., Dryden, M.W., Guptill, L.C., Iazbik, C., Kania, S.A., Lathan, P., Little, S.E., Roy, A., Sayler, K.A., Stillman, B.A., Welles, E.G., Wolfson, W., Yabsley, M.J., 2012. Seroprevalence of Ehrlichia canis, Ehrlichia chaffeensis, and Ehrlichia ewingii in dogs in North America. Parasit. Vectors 5, 29. Bowman, D., Little, S.E., Lorentzen, L., Shields, J., Sullivan, M.P., Carlin, E.P., 2009. Prevalence and geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum in dogs in the United States: results of a national clinic-based serologic survey. Vet. Parasitol. 160, 138–148. Centers for Disease Control and Prevention, 2014. http://www.cdc.gov/ ticks/geographic_distribution.html (accessed 06.24.14). Chandrashekar, R., Mainville, C.A., Beall, M.J., O’Connor, T., Eberts, M.D., Alleman, A.R., Gaunt, S.D., Breitschwerdt, E.B., 2010. Performance of a commercially available in-clinic ELISA for the detection of antibodies against Anaplasma phagocytophilum, Ehrlichia canis, and Borrelia burgdorferi and Dirofilaria immitis antigen in dogs. Am. J. Vet. Res. 71, 1443–1450. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit. Vectors 3, 26. Duncan, A.W., Correa, M.T., Levine, J.F., Breitschwerdt, E.B., 2005. The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic states. Vector Borne Zoonotic Dis. 5, 101–109. Ewing, S.A., Dawson, J.E., Kocan, A.A., Barker, R.W., Warner, C.K., Panciera, R.J., Fox, J.C., Kocan, K.M., Blouin, E.F., 1995. Experimental transmission of Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) among white-
tailed deer by Amblyomma americanum (Acari: Ixodidae). J. Med. Entomol. 32, 368–374. Gaunt, S., Beall, M., Stillman, B., Lorentzen, L., Diniz, P., Chandrashekar, R., Breitschwerdt, E., 2010. Experimental infection and co-infection of dogs with Anaplasma platys and Ehrlichia canis: hematologic, serologic, and molecular findings. Parasit. Vectors 3, 33. Hegarty, B.C., Maggi, R.G., Koskinen, P., Beall, M.J., Eberts, M., Chandrashekar, R., Breitschwerdt, E.B., 2012. Ehrlichia muris infection in a dog from Minnesota. J. Vet. Intern. Med. 26, 1217–1220. Johnson, E.M., Ewing, S.A., Barker, R.W., Fox, J.C., Crow, D.W., Kocan, K.M., 1998. Experimental transmission of Ehrlichia canis (Rickettsiales: Ehrllichieae) by Dermacentor variabilis (Acari: Ixodidae). Vet. Parasitol. 74, 277–288. Kordick, S.K., Breitschwerdt, E.B., Hegarty, B.C., Southwick, K.L., Colitz, C.M., Hancock, S.I., Bradley, J.M., Rumbough, R., McPherson, J.T., MacCormack, J.N., 1999. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J. Clin. Microbiol. 37, 2631–2638. Little, S.E., 2010. Ehrlichiosis and anaplasmosis in dogs and cats. Vet. Clin. North Am. Small Anim. Pract. 40, 1121–1140. Little, S.E., O’Connor, T.P., Hempstead, J., Saucier, J., Reichard, M.V., Meinkoth, K., Meinkoth, J.H., Andrews, B., Ullom, S., Ewing, S.A., Chandrashekar, R., 2010. Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States. Vet. Parasitol. 172, 355–360. Murphy, G.L., Ewing, S.A., Whitworth, L.C., Fox, J.C., Kocan, A.A., 1998. A molecular and serologic survey of Ehrlichia canis, E. chaffeensis, and E. ewingii in dogs and ticks from Oklahoma. Vet. Parasitol. 79, 325–339. Ndip, L.M., Ndip, R.N., Ndive, V.E., Awuh, J.A., Walker, D.H., McBride, J.W., 2007. Ehrlichia species in Rhipicephalus sanguineus ticks in Cameroon. Vector Borne Zoonotic Dis. 7, 221–227. Nicholson, W.L., Allen, K.E., McQuiston, J.H., Breitschwerdt, E.B., Little, S.E., 2010. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol. 26, 205–212. Pritt, B.S., Sloan, L.M., Johnson, D.K., Munderloh, U.G., Paskewitz, S.M., McElroy, K.M., McFadden, J.D., Binnicker, M.J., Neitzel, D.F., Liu, G., Nicholson, W.L., Nelson, C.M., Franson, J.J., Martin, S.A., Cunningham, S.A., Steward, C.R., Bogumill, K., Bjorgaard, M.E., Davis, J.P., McQuiston, J.H., Warshauer, D.M., Wilhelm, M.P., Patel, R., Trivedi, V.A., Eremeeva, M.E., 2011. Emergence of a new pathogenic Ehrlichia species, Wisconsin and Minnesota, 2009. N. Engl. J. Med. 365, 422–429. Qurollo, B.A., Davenport, A.C., Sherbert, B.M., Grindem, C.B., Birkenheuer, A.J., Breitschwerdt, E.B., 2013. Infection with Panola Mountain Ehrlichia sp. in a dog with atypical lymphocytes and clonal T-cell expansion. J. Vet. Intern. Med. 27, 1251–1255. Qurollo, B.A., Chandrashekar, R., Hegarty, B.C., Beall, M.J., Stillman, B., Liu, J., Thatcher, B., Pultorak, B., Comyn, A., Bretischwerdt, E.B., 2014. A descriptive analysis of clinical manifestations in dogs with tick-borne pathogen co-infections and co-exposures. J. Vet. Intern. Med. 28, 976–1134. Ristic, M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K., Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun. 6, 226–231. Yabsley, M.J., Adams, D.S., O’Connor, T.P., Chandrashekar, R., Little, S.E., 2011. Experimental primary and secondary infections of domestic dogs with Ehrlichia ewingii. Vet. Microbiol. 150, 315–321. Yabsley, M.J., Loftis, A.D., Little, S.E., 2008. Natural and experimental infection of white-tailed deer (Odocoileus virginianus) from the United States with an Ehrlichia sp. closely related to Ehrlichia ruminantium. J. Wildl. Dis. 44, 381–387.
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